Protocol for quantitative evaluation of the impact of paracrine senescence on cellular reprogramming in cultured cells and mouse models

Summary We present a protocol to evaluate the impact of senescence secretome on reprogramming to pluripotency using both cellular and mouse models. First, we describe the in vitro reprogramming procedure using conditioned medium derived from senescent cells. Next, to explore the impact of senescence on in vivo reprogramming, we detail the steps to identify senescent and reprogrammed cells in mouse skeletal muscle, followed by semi-automatic quantification. This protocol can be used to study the effect of paracrine senescence on cellular plasticity. For complete details on the use and execution of this protocol, please refer to von Joest et al. (2022).1


Highlights
Protocol for studying the effect of paracrine senescence on cellular reprogramming Assessment of in vitro reprogramming in cultured cells and in vivo reprogramming in mice Steps for co-staining senescent cells with cell-identity markers in tissue Semi-automatic quantification of SAb-Gal+ senescent cells in tissue sections

SUMMARY
We present a protocol to evaluate the impact of senescence secretome on reprogramming to pluripotency using both cellular and mouse models. First, we describe the in vitro reprogramming procedure using conditioned medium derived from senescent cells. Next, to explore the impact of senescence on in vivo reprogramming, we detail the steps to identify senescent and reprogrammed cells in mouse skeletal muscle, followed by semi-automatic quantification. This protocol can be used to study the effect of paracrine senescence on cellular plasticity. For complete details on the use and execution of this protocol, please refer to von Joest et al. (2022). 1

BEFORE YOU BEGIN
Senescence regulates cellular plasticity in cell-autonomous and non-cell-autonomous manners. The senescence secretome, known as the senescence-associated secretory phenotype (SASP), is crucial for non-cell autonomous functions of senescence. Notably, SASP composition is stress-and cell-type dependent. To identify specific SASP factors important for promoting cellular plasticity, we use a reprogrammable mouse model (i4F), Rosa26-rtTA, and TetO-OSKM. Reprogramming can be induced by the addition of doxycycline (DOX). This protocol is divided into in vitro and in vivo sections. First, we describe the conditioned medium (CM) system to compare the effects of stress-dependent paracrine senescence on in vitro reprogramming. Second, to explore the impact of paracrine senescence on in vivo reprogramming, we detail the specific steps of the co-identification of senescence-associated b-galactosidase (SA-b-Gal+) and reprogramming cells (Nanog+) in the tibialis anterior (TA) muscle of reprogrammable mice. Previously, we've shown the combination of injury and DOX administration results in senescence induction and reprogramming of TAs. 2 Finally, we describe a semi-automatic image analysis program for quantifying the number of senescent cells in vivo.
The in vitro part of the protocol can be applied to any cellular system of cell fate conversion, with some modifications to the CM supplementation step (see conditioned iPSC medium). The in vivo part of the protocol can be applied to any tissue from i4F mice, with some adjustments in SAb-Gal staining incubation time, to animals of both sexes and of different ages, from adulthood to age. Moreover, this part of the protocol can facilitate the identification and quantification of senescent cells in physiological and pathological processes, beyond reprogramming.  Note: Do not remove the medium from the culture plate, simply add the Fugene6/DNA mix to the medium dropwise with the P1000 pipette, distributing it around the plate.

Reagent
Stock concentration Final concentration Amount The fixation solution can be stored at 4 C up to 6 months. a. Collect the medium from transfected HEK293T cells and replace with 10 mL of fresh MEF medium. b. Centrifuge each medium separately at 2,000 g for 5 min at RT to remove cell debris. c. Filter the supernatants with 0.45 mm filters separately. d. Add Polybrene (8 mg/mL final concentration). e. Repeat the infection one more time after 8-10 h. 4. Day 4: Remove the medium, and refresh with 10 mL MEF medium. 5. Day 5:

Reagent
a. Trypsinize the cells from each plate and reseed them at the 5 3 10 5 cells/P100 plate with MEF medium containing Puromycin (1 mg/mL). b. Plate non-infected WT MEFs at the same condition as the negative control for Puromycin selection. 6. Day 7: Refresh the medium with MEF medium containing 1 mg/mL Puromycin. 7. Day 9: When there is no MEFs visible from the negative control plate (usually after 96 h), remove the medium and replace with fresh conditioned MEF medium without Puromycin for conditioned medium collection.
Note: At this point, if you perform the SA-b-gal assay, OIS-senescent MEFs will be stained in blue while non-senescent MEFs will not ( Figure 1A).

Timing: 6 days
This section describes how to collect the conditioned medium from senescent cells.
8. After two days in conditioned MEF medium, collect the culture medium from both conditions, and replenish fresh conditioned MEF medium for next rounds of collection.
Note: Senescent conditioned medium can be collected three times (48 h interval).
Note: Make sure to collect the conditioned medium from the non-senescent counterpart (PIGinfected plate), which should be used as the control for senescent conditioned medium.
9. For the collected culture medium, centrifuge for 5 min at 500 g at RT. 10. Filter the supernatant with a 0.20 mm sterile filter, and store the filtered medium in several aliquots at À20 C.
Pause point: Filtered conditional medium can be stored at À20 C for up to 1 month.
CRITICAL: Repeated freeze/thaw cycles could greatly diminish the effects of the conditional medium. Therefore, we recommend to aliquot conditional media in proper aliquoted volumes depending on the experimental requirements to avoid repeated freeze/thaw cycles.
11. If separation of the exosomes from the soluble fraction is desired: a. Centrifuge the conditioned medium at 12,000 g for 30 min at 4 C, and retain the supernatant and discard the pellet. b. Ultracentrifuge the supernatant at 100,000 g for 3 h at 4 C, and separate the supernatant from the pellet. c. Store the soluble fraction at À20 C in aliquoted volumes. d. Wash once by resuspending the exosomes in 1 mL PBS. e. Ultracentrifuge at 100,000 g for 3 h at 4 C, discard the supernatant, and keep the pellet. f. Resuspend the pellet in 20-50 mL PBS and store it at À20 C.
Pause point: Exosomes in PBS can be stored for up to 1 month at À20 C, or alternatively for up to 1 year at À80 C, avoiding repeated freeze/thaw cycles.

Induction of reprogramming via senescent conditioned medium
Timing: 10-14 days This section describes how to induce reprogramming in i4F MEFs using senescent conditioned medium.
Note: The seeding density of the i4F MEFs is highly dependent on their reprogramming capacity. Therefore, we recommend testing the reprogramming efficiency of individual i4F MEF before this experiment.
13. The following day, remove MEF medium, and replace it with conditioned iPSC medium freshly supplemented with DOX (1 mg/mL).
CRITICAL: DOX must be added freshly to iPSC medium because its stability rapidly decreases at 37 C. We recommend changing iPSC medium supplemented with DOX every two days maximum.
14. Change the conditioned iPSC medium supplemented with DOX (1 mg/mL) every day. 15. After 3 days, change the conditioned iPSC medium to normal iPSC medium freshly supplemented with DOX (1 mg/mL). 16. Renew the medium every 2 days.
Note: It takes approximately 10-14 days to generate iPSC colonies using a reprogrammable MEFs system. The experimental length and period of conditioned iPSC medium treatment may vary depending on the reprogramming system implemented. DOX concentration may also be an important factor in reprogramming efficiency for different cell types; thus, we recommend testing different DOX concentrations.
PART II: In vivo identification of senescent and pluripotent cells: IHC Co-staining with SAb-gal staining Tissue collection and procession

Timing: 1 day
This section describes how to generate cryosections from injured murine skeletal muscle.
18. 10 days prior of the sample collection, inject cardiotoxin (CTX) into tibialis anterior (TA) muscle of mice of both sex (2-month-old, C57/B6) to injury muscle as previously described. 6 Note: Make sure to inject PBS in the TA of the same mouse as non-injured negative control.
19. Collect TAs at 10 days post-injury: a. Place a small amount of tragacanth gum on a slice of cork. b. Extract both TA muscles as previously described. 7 c. To ensure the transverse sections, insert the distal tendon of the TA muscle (1/4 part) into the tragacanth gum and freeze directly in liquid nitrogen cooled isopentane for 40 s as previously described. 7 Note: Make sure the TA muscle is in a perpendicular position and in the center of the cork.  25. Refresh the staining solution after 24 h. 26. Check the slices using an inverted cell culture microscope (OLYMPUS CKX41). Once the blue color is visible (Figure 2A), quickly wash the slices three times with PBS.
Note: The staining duration is tissue section dependent (tissue origin and section thickness). It needs to be optimized first and should be kept consistent for the samples from the same experiment. For example, for the muscle samples, we incubate 10 mm sections for 48 h. For other tissues, the staining need to be optimized further. In general, thicker sections need shorter incubation time than thinner sections. For example, sections from kidney and lung need shorter incubation time than muscle.

Timing: 24 h
This section describes how to perform IHC staining targeting Nanog (or F4/80) on muscle cryosections. These procedures are performed following SA-b-Gal staining.
27. Quickly wash the slices three times with PBS and put the slides in 4% PFA in PBS for post-fixation for 15 min at RT.
CRITICAL: Post-fixation cannot be longer than 15 min.
28. Quickly wash the slides three times with PBS and place them in a humid dark chamber (Figure 2B).  CRITICAL: All the primary antibodies dilution and the duration of DAB staining needs to be optimized first.
Note: This protocol can be used for other antibodies, such as F4/80.

PART III: Semi-automatic quantification of senescent cells based on SA-b-Gal staining.
The entire procedure is explained in Methods video S1 (or on YouTube with subtitles: https://www. youtube.com/watch?v=BHaThFfpkRY) to guide you in running Showblue. a. On macOS, right-click on the ''showblue-main'' folder and select ''new terminal at folder''. The Terminal will appear, drag the file named show_all_fig_para.sh into the Terminal, and press enter. b. On Windows, double click on the start_envrionment.bat file. A window will open, drag into it the file named show_all_windows_fig_para.bat, and press enter.

Installation of Showblue and its dependencies
Note: Showblue will quantify the images automatically. When the quantification is finished, a new file named ''data.txt'' and a new folder named ''process'' will be generated in the ''showblue-main'' folder.
Note: Showblue will also set up all text parameters (cfg_bk.txt, cfg_in.txt, and cfg_pt.txt) in the ''cfg'' folder automatically according to the labeled images in the ''cfg_fig6'' folder. If the results are not satisfactory, the text parameters can be further adjusted manually (step 53).  52. Check the quantification results in the ''process'' folder ( Figure 5).
Note: The image labeled ''blue'' displays the number of detected SA-b-Gal+ cells (top left) and the pixels 2 surface area of the muscle section (top right) ( Figures 5A and 5B). The image labeled ''bound'' displays the muscle section boundaries that are excluded from the analysis ( Figure 5C). And the image labeled ''hull'' displays the area that is analyzed ( Figure 5D).
53. Configure the Showblue settings manually if automatic settings are not satisfactory, or if you want to further optimize the detection. The text parameters are located in the folder named ''cfg'': a. cfg_alpha.txt: the ''alpha value'' is used to adjust the sensitivity of tissue boundary detection. By default, the alpha value is set to 1. The larger the alpha value, the higher the sensitivity. b. cfg_bk.txt: it describes the ''mean color value G the standard deviations (SD)'' of the points outside the tissue boundary (background). c. cfg_in.txt: it describes the ''mean color value G SD'' of all the points inside the tissue boundary. d. cfg_pt.txt: it describes the ''mean color value G SD'' of the points of interest inside the tissue boundary.
Note: The color value is defined using an RGB color model. Each of the primary additive colors of red, green, and blue is assigned a value in the range of 0-255.
e. cfg_shape.txt: the ''shape value'' is to remove colored cells whose shape is not of interest. The value is set ranging from 0-1. If the value is 0, all colored items are counted as cells, including dye contaminations. When the value approaches 1, the selected cells are closer to the round shape and the counting sensitivity is low.  Note: depending on the computer power, and the number and size of images that need to be quantified, this step can vary considerably over time.

EXPECTED OUTCOMES
The outcome of PART I is a reliable and consistent collection of senescent MEF conditional medium to study the effect of SASP on cellular reprogramming in vitro. 1 The composition of the SASP is highly heterogeneous and can have a distinct effect on reprogramming efficiency. Therefore, it is crucial to understand how SASP composition affects different processes and how cellular reprogramming is initiated in a paracrine manner.
Here, we provide a feasible protocol for identifying and investigating the potential molecular mechanisms underlying these two processes. A successful and efficient reprogramming is characterized by the formation of $20 iPSC colonies per well of a 6-well plate within 14 days ( Figure 1C) and quantified using Fiji.
Co-staining of SA-b-Gal with other markers in tissue samples has been difficult owing to incompatible fixation and staining conditions. Here in PART II, we present a robust protocol to simultaneously identify senescent (SA-b-Gal + ) and reprogrammed (Nanog + ) cells in the same TA muscle sample ll OPEN ACCESS collected from reprogrammable mice. Successful co-staining is shown in Figure 2C, with SA-b-Gal staining in blue and IHC staining in brown. No signals were detected in the negative control (noninjured muscle for SA-b-Gal and the section without 1 st antibody for Nanog). This protocol can be used to determine the identity of senescent cells in vivo, when combined with cell identity markers. For example, macrophages have an increased lysosomal content and can be positive for SA-b-Gal staining. To determine whether SA-b-Gal + cells were macrophages, we co-stained for SA-b-Gal and F4/80, a macrophage marker ( Figure 2D). Interestingly, although many cells were positive for both markers, we also found that the cells were only positive for SA-b-Gal or F4/80, suggesting a heterogeneous senescent population ( Figure 2D).
Quantifying SA-b-Gal-positive cells in vivo is time consuming and subject to user experience. We present a semi-automatic plug-in to facilitate this process. The false-positive or false-negative rates should not exceed 10% ( Figure 5B), and calculation of the area should be performed automatically ( Figure 5D). Although this program does not accurately count absolute numbers, the relative numbers are reproducible. Therefore, it is crucial to quantify samples from different groups simultaneously. It usually takes 1-2 h to set up the preferences and an hour to collect data with this program. Therefore, it provides an efficient and consistent alternative to the conventional manual counting methods.

QUANTIFICATION AND STATISTICAL ANALYSIS
Quantification and statistical analysis should be performed by a blinded operator to avoid bias. Statistical analyses were performed using the GraphPad Prism v9 software.

LIMITATIONS
The protocol described here aims to study the impact of paracrine senescence on cellular plasticity in the context of cellular reprogramming to pluripotency. Therefore, a reprogrammable mouse model and an MEF system are used to allow robust and reliable reprogramming induction. The link between senescence and plasticity has also been demonstrated in other physiological and pathological conditions. Therefore, the in vitro conditioned medium method can be modified to study how non-cell-autonomous senescence affects cellular plasticity beyond the reprogramming context. The method used to identify and quantify senescent cells can be applied to characterize in vivo senescence.
For in vitro experiments, the protocol is performed using conditioned medium derived from senescent MEFs only. Paracrine senescence is heterogeneous, which is stress-and cell-type dependent 8 ; thus, conditioned medium from other cell types could be different from MEFs.
It is possible that senescent conditioned medium from certain cell types might prevent reprogramming. We encourage you to report such observations and studies to better understand the link between senescence and cellular reprogramming and plasticity.
In vitro reprogramming is performed using i4F MEFs only. Reprogramming kinetic and efficiency can vary greatly depending on the cell type and reprogramming methods. Therefore, the initial seeding density of the reprogramming cells, DOX concentration, and treatment length of the conditioned iPSCs medium should be determined first.
To ensure accurate quantification, well-preserved tissue sections and high-quality of the SA-b-Gal staining are crucial. In addition, only the samples processed, stained, and analyzed in the same patch can be compared.

Problem 1
You don't obtain any iPSCs colony after applying conditioned medium on i4F MEFs (step 16, Figure 1B). Potential solution Make sure the conditioned medium has not undergone more than one freeze/thaw cycle. Precipitates can form and alter conditional media composition, resulting in a poor reprogramming efficiency. We recommend to aliquot your conditional medium in small volumes. Moreover, the initial seeding density of the reprogramming cells might be too low. The reprogramming efficiency might vary significantly from one embryo to another, and by using different reprogramming systems. Therefore, the seeding density should be determined prior.

Problem 2
You don't see the blue developed after 48 h SA-b-Gal (step 26, Figure 2A).

Potential solution
As described above, make sure pH is 5.5 for the PBS and all the other components for the staining solution are clean without contaminations. For the different tissue and the thickness of the sections, the incubation time is different. If the staining doesn't work, the incubation time should be optimized. It is crucial to include positive and negative control for each experiment.

Problem 3
Error for installation of library cv2 (step 44).

Potential solution
Sometimes, there is a problem in installing cv2 with the error information as ''Could not build wheels for opencv-python which use PEP 517 and cannot be installed directly''. In such a case, it is necessary to run the command ''pip install -upgrade pip setuptools wheel'' before installing of cv2.

RESOURCE AVAILABILITY
Lead contact Further information and requests for resources and reagents should be directed to and will be fulfilled by the lead contact, Han Li (han.li@pasteur.fr).

Materials availability
This study did not generate new unique reagents.